Cyclen-Based Side-Chain Homopolymer Self-Assembly with Plasmid DNA: Protection of DNA from Enzymatic...

Cyclen-Based Side-Chain Homopolymer Self-Assembly with Plasmid DNA:Protection of DNA from Enzymatic Degradation

by Kun Lia), Na Wanga), Fan Yanga), Zhong-Wei Zhangb), Li-Hong Zhoua), Shan-Yong Chena),Hong-Hui Lin*b), Yun-Fei Tianc), and Xiao-Qi Yu*a)d)

a) Department of Chemistry, Key Laboratory of Green Chemistry and Technology (Ministry ofEducation), Sichuan University, Chengdu 610064, P. R. China

b) Key Laboratory of Bio-resources and Eco-environment (Ministry of Education), College of LifeSciences, Sichuan University, Chengdu 610064, P. R. China

c) Analytical and Testing Center, Sichuan University, Chengdu, 610064, P. R. Chinad) State Key Laboratory of Biotherapy, West China Hospital, West China Medical School, Sichuan

University, Chengdu 610041, P. R. China(fax: þ86 28 85415886; e-mail: [email protected])

In this study, a 1,4,7,10-tetraazacyclododecane (cyclen)-based side-chain homopolymer wasdeveloped for the first time; this polymer can self-assembly with plasmid DNA to form polyelectrolytecomplexes (polyplex), which can protect DNA from enzymatic degradation. Moreover, the polyplex candisassembly and release free DNA when NaCl solution is added to this system. Scanning electronmicroscopy (SEM) and atomic force microscopy (AFM) are used for imaging of the surface structure ofthe polyplex, and results indicated that the polyplex structures respond to the polymer concentration.Circular dichroism (CD) spectrum suggested that the DNA configuration in the polyplex was retained.

1. Introduction. – DNA Protection has been regarded as an important prerequisitein gene delivery; the design of materials that provide spatial or temporal protection ofDNA via self-assembly and disassembly under physiological conditions is an activeresearch area [1] [2]. Some methods have been reported for DNA protection, such asdeveloping amino-modified silica nanoparticles, preparing phosphorylcholine basedcopolymers, or multilayered polyelectrolyte films, and forming ultrasound-responsivemicrobuttles [3– 6]. In recent years, interest has been focused on the development ofpolycation/DNA complexes with noncovalent interactions, which is regarded as anefficient method to prevent DNA from enzymes degradation [7 –9]. Additionally, usingsynthetic polycations for self-assembly with DNA facilitates adjustment the bindingability by selecting respective monomers or optimum composition of copolymers tomodify the polymer structure [10]. Polycation polymers, such as polyethylenimine,polylysine, and low-molecular-weight dendrons, have been widely used to condenseDNA and further deliver genetic information into cells [11 – 15]. For example,polyplexes formed by polycations with primary amines efficiently protect DNA fromrelease and degradation during transport and release DNA in the cytoplasm or nucleus[10]. Janout et al. reported molecular umbrella-spermine can significantly enhanceDNA-binding ability [16]; and, more recently, Borodina et al. developed a self-degrading microcapsules to control release DNA [17].

CHEMISTRY & BIODIVERSITY – Vol. 6 (2009)754

� 2009 Verlag Helvetica Chimica Acta AG, Z�rich

Cyclen (¼1,4,7,10-tetraazacyclododecane), as one of the most common macro-cyclic polyamine compounds, is an important complexing agent for cations, anions, andneutral molecules, and has been widely used in molecular recognition, chemicalnucleases, and sensors [18 – 21]. Some cyclen complexes, such as the conjugate ofuracil –cyclen ZnII complex, showed good cleavage ability for DNA [22] [23]. However,research has also been conducted on the cyclen-based polymer interaction with DNA.Hwang et al. reported a cyclen-CuII based copolymer (CuII-cyclen-pHEMA) as amaterial generating NO from naturally occurring RSNO species [24]. Suh and co-workers attached CuII – cyclen complex to poly[(chloromethyl)styrene-co-divinylben-zene] as an efficient artificial enzyme for DNA cleavage [25 –27]. To utilize more local-concentrated protonated amines of cyclen, we successfully synthesized a cyclen-basedside-chain cationic polymer and investigated its interaction with DNA.

Herein, we report, for the first time, preparation of a cyclen-based side-chain polymeras a new potential DNA-protection agent possessing high affinity and retaining the DNAconfiguration. The cyclen-based polymer can self-assembly with plasmid DNA-formingpolyplex, which then protect DNA strands from enzymatic degradation.

2. Results and Discussion. – 2.1. Preparation of the Cyclen-Based Side-ChainHomopolymer. A cyclen-based side-chain homopolymer (poly[1-(N-methacryloyl-2-aminoacetyl)-1,4,7,10-tetraazacyclododecane], PMAC; methacryloyl¼2-methylprop-2-enoyl) was synthesized by polymerization of 1-(N-methacryl-2-aminoacetyl)-1,4,7,10-tetraazacyclododecane trifluoroacetic acid salt (5, MAC ·3 TFA) initiated with(NH4)2S2O8/NaHSO3 (Scheme). The monomer 5 was obtained by coupling ofTris(Boc)-Cyclen and Fmoc-Gly, then removal of the protecting group (Fmoc¼ (9H-fluoren-9-yl)methoxycarbonyl) and acylation with methacryloyl (¼2-methylprop-2-anoyl) chloride, and finally removal of the Boc protecting group by CF3COOH.

2.2. Polyplex Formation of PMAC (6) with DNA. Electrophoretic mobility shiftassay was performed to investigate the interaction of PMAC (6) with plasmid DNA.Polyplexes were obtained by mixing aqueous solutions of 6 and DNA at various N/Pratios between 1.18 and 67.60, loaded into the gel, and submitted to electrophoresis. Asshown in Fig. 1, the electrophoretic mobility of plasmid DNA was retarded bycondensation with PMAC (6) at N/P ratios 6.77– 67.60, clearly demonstrating that thepolymer interacted electrostatically with DNA and formed stable complexes. However,free DNA band was found in the gel at the N/P ratio 1.18, which implies that theplasmid DNA had weak interaction with 6 and could not totally encased by 6.

The shape and particle size distributions of the polymer/DNA complex at variouscharge ratios were examined by SEM. The side-chain polymer preferred to form smallparticles with a diameter in the range of 2 –5 mm without DNA (Fig. 2, a), while thepolymer would assembly with this �anionic polymer� to form PMAC (6)/DNA polyplexthrough electrostatic interactions in the presence of DNA. As shown in Fig. 2,b, someDNA were attached on the surface of polymer, some connected two or more polymermolecules to form straight-line macromolecules, and others might cross-link thepolymer molecules, which made the polyplex diameter range from 10 to 30 mm.However, if the polyplex concentration increases, fibrillar network morphology wouldbe found (Fig. 2, c); these results are in agreement with the results reported by Dawnand Nandi group [28].

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Scheme. Synthesis of Poly[1-(N-methacryloyl-2-aminoacetyl)]-1,4,7,10-tetraazacyclododecane (6, PMAC)

Fig. 1. Agarose gel-electrophoretic DNA-binding shift assay for PMAC at various N/P ratios. Lane 1:plasmid DNA (7 mg/ml), and Lanes 2–6: polyplex formulations with different N/P ratios (1.18, 6.77,16.90, 33.80, and 67.60, resp.). All samples were run on a 0.8% agarose gel and stained with ethidium

bromide.

To further study DNA-binding characteristics of the PMAC (6), atomic forcemicroscopy (AFM) was used to determine the morphological changes in the shape ofDNA after interaction with 6. When DNA isolated was imaged, extended DNA strandswere visible (Fig. 3,a). The diameter of DNA chain was observed around 50 nm. At anN/P ratio of 6.77, various types of particles were visible, most of them were smallglobules, but a few large globules were also seen. This indicated that the DNAunderwent assembly with the polymer or was enclosed by the polymer, also confirmingthe assembly mode we proposed in the SEM, i.e., the large globules were assumed to beformed between the small polyplexes via DNA bridges. Fig. 3,b displays globules with adiameter around 380 nm; the AFM images give an obvious contrast before and afterthe assembling.


Fig. 2. SEM Images of PMAC and PMAC/DNA polyplex. a) PMAC alone; b) polyplex N/P 6.77; c)polyplex N/P 67.60.

Fig. 3. AFM Images of the DNA (a) and PMAC/DNA polyplex (b) at N/P ratio of 6.77

2.3. Protection of DNA against DNase I. It is essential for DNA assembly with thecomplex to be strong enough to allow protection against biological enzymes. Theprotection effect of polyplex against DNase I degradation was investigated by gelelectrophoresis, and the result is shown in Fig. 4. When plasmid DNA was incubatedwith DNase I in the absence of PMAC (6), supercoiled DNA was completely degradedafter 1 h (Fig. 4, Lane 2); however, when 6 was added to the solution, no degradation ofDNA was observed (Fig. 4, Lane 3). The enzyme-degradation assay showed thatPMAC (6) polymer provided effective DNA protection against enzymatic degradation.

2.4. DNA-Binding Ability of PMAC (6) and DNA Release from the Polyplex. Thebinding ability of the side-chain polymer to DNA was studied by using an ethidiumbromide (EB) displacement assay. This method is commonly used to study the bindingof polyammonium compounds to DNA [29]. According to the Stern – Volmer equation[30]: F0/F¼1þKsv[Q], the binding constant is 4.2�103, indicating 6 has a moderatebinding ability (Fig. 5). Therefore, the 6/DNA polyplex can disassemble in thepresence of sodium polyacrylate (NaPAA) or NaCl, and release free DNA (Fig. 6).Additionally, circular dichroism (CD) spectrum was used to investigate the config-uration of DNA before and after 6 was added (Fig. 7), and the results suggested theretention of the configuration.

2.5. Analysis of Buffer Capacity by pH Titration. Potentiometric titration curveswere shown in Fig. 8 (Insert). Buffer capacity (b) was calculated from these data usingthe equation [31– 33]: b¼d[HCl]/d(pH). The results showed that the largest bufferingcapacity of PMAC (6) appeared at pH values above 8.0 (Fig. 8), which indicated that alarge quantity of secondary amines existed in 6. These results in agreement with otherpolyamines reported by other groups [31] [32]. At pH 7.0, around 500 ml 0.1n HCl wasneeded, the Hþ amount was well consistent with the amine quantity of cyclen inPMAC, which suggested that only the NH in cyclen unit were protonated.

3. Conclusions. – In summary, a cyclen-based side-chain polymer with high DNA-binding activity was developed. The cyclen-based polymer can self-assembly with


Fig. 4. Protection assay of PMAC/DNA by DNase I. Lane 1: DNA alone (7 mg/ml), Lane 2: DNAþDNase I (2U); Lane 3: DNAþDNase I (2U)þPMAC (14.3 mg/l), Lane 4: DNAþPMAC (14.3 mg/l),

Lane 5: DNA marker (lDNA is cleavaged by Hind III).


Fig. 5. a) Fluorescence quenching of DNA-EB system by addition PMAC soln. b) Linear fitting accordingthe relative intensity (F0/F) in different concentrations of PMAC.

Fig. 6. a) Lane 1: DNA alone (7 mg/ml), Lanes 2–6: DNAþPMAC (14.3 mg/l)þNaPMAA 0, 0.14,0.28, 0.43, 0.57 g/l. b) Lane 1: DNA alone (7 mg/ml), Lanes 2–5: DNAþPMAC (14.3 mg/l)þNaCl soln.

0, 0.85, 1.25, 1.70 mol/l.

Fig. 7. CD Spectra of DNA alone and PMAC/DNA polyplex

plasmid DNA to form polyplex, retaining DNA primary configuration. SEM Images,however, indicate that assembly mode is responding to the polymer concentration andformation of fibrillar network morphology in high polyplex concentration. Moreinterestingly, the PMAC (6)/DNA polyplex can efficiently protect DNA against DNaseI. Also, the polyplex can undergo disassembly by NaPAA or NaCl. Formingtemporarily stable DNA polyplex and protecting DNA from enzyme degradation arethe two important key steps for gene delivery. Thus, PMAC (6) is expected to be used ingene therapy.

Experimental Part

1. General. All chemicals and reagents were obtained commercially and were used as received. Anh.MeCN, THF, CHCl3, and CH2Cl2 were dried and purified under N2 by standard methods and weredistilled immediately before use. All aq. solns. were prepared from deionized or dist. H2O. Electro-phoresis-grade agarose, ethidium bromide (EB), bromophenol blue, DNase I, plasmid DNA (pUC19),salmon sperm DNA, and calf thymus DNA (CT DNA) were purchased from Takara Biotechnologycompany. DNA-Loading buffer contained 0.25% (w/v) bromophenol blue in a 40% sucrose soln. 1,4,7-Tris[(tert-butoxy)carbonyl]-1,4,7,10-tetraazacyclododecance (3Boc-cyclen, 1) was prepared according tothe literature [34] [35]. Prep. gel-permeation chromatography: Waters instrument incorporating Shodexcolumns (OHPAK KB-803); calibrated using polyethylene standard. Circular dichroism (CD) spectra:JASCO-500C spectropolarimeter. IR Spectra: Shimadzu FTIR-4200 spectrometer as KBr pellets.1H-NMR Spectra: Varian INOVA-400 spectrometer; d in ppm referenced to residual solvent peaks orinternal Me4Si. HR-ESI-MS: Finnigan LCQDECA and a Bruker Daltonics Bio TOF mass spectrometer,resp. Electrophoresis apparatus was a Biomeans Stack II electrophoresis system, PPSV-010. Bands werevisualized by UV light and photographed using a gel-documentation system by the estimation of theintensity of the DNA bands, recorded on an Olympus Grab-IT 2.0 annotating image computer system.

2. Preparation of 1,4,7-Tris[(tert-Butoxy)carbonyl]-10-(2-{[(9H-fluoren-9-yl)methoxycarbonyl]ami-no}acetyl)-1,4,7,10-tetraazacyclododecane (2). To a stirred soln. of Fmoc-Gly (1.38 g, 4.66 mmol) in


Fig. 8. Buffer capacity (b) of PMAC solution (1.0 mg/ml) as function of pH. Inset: pH Titration of PMACand NaCl solution (1 mm).

CH2Cl2 (60 ml) in an ice-water bath, 1 (2.0 g, 4.24 mmol) was added. DCC (N,N’-Dicyclohexylcarbo-diimide; 0.96 g, 1.1 mmol), dissolved in CH2Cl2 (20 ml), was dropped slowly to the soln. The soln. wasstirred at 08 for 1 h, and stirring was continued at r.t. overnight, then the mixture was filtered to removethe urea side-product. The solvent was removed under reduced pressure. The obtained solid wasdissolved in AcOEt (30 ml) and freezed for 5 h. After the urea-side product was filtered, the solvent wasremoved under reduced pressure again. The crude product was purified by column chromatography (CC;SiO2; AcOEt/petroleum ether (PE) 1 : 1) to yield pure 2 (3.0 g, 92%). White solid. M.p. 76–788. 1H-NMR(400 MHz, CDCl3): 7.76 (d, J¼7.2, 2 H, Fmoc-CH); 7.61 (d, J¼7.6, 2 CH (Fmoc)); 7.40 (t, J¼6.0, 2 CH(Fmoc)); 7.29 –7.33 (m, 2 CH (Fmoc)); 4.36 (d, J¼7.5, CH2O); 4.23 (t, J¼7.6, CHCH2); 4.03 (d, J¼5.4,CH2NH); 3.40–3.48 (m, 8 CH2 (cyclen)); 1.46–1.48 (m, 9 Me (Boc)). ESI-MS: 774.4 ([MþNa]þ ).

3. Preparation of 1-(2-Aminoacetyl)-4,7,10-tris[(tert-butoxy)carbonyl]-1,4,7,10-tetraazacyclodode-cane (3). To a stirred soln. of 2 (2.96 g, 4.61 mmol) in CH2Cl2 (75 ml) under N2 at r.t. was slowly addedpiperidine (15 ml). The soln. was stirred for 2 h, and the solvent was removed under reduced pressure.Then, the crude product was dissolved in CH2Cl2 (120 ml) and washed with H2O (3�50 ml), 5% citricacid (30 ml), 1n aq. NaOH (30 ml), and H2O (50 ml). The org. phase was dried (Na2SO4) and filtered.The solvent was removed under reduced pressure. The crude product was purified by CC (SiO2; CHCl3/AcOEt/MeOH 2 : 2 : 1) to yield pure 3 (1.43 g, 58%). White solid. M.p. 94–958. 1H-NMR (400 MHz,CDCl3): 3.38–3.55 (m, 8 CH2 (cyclen)); 1.46–1.48 (m, 9 Me (Boc)). ESI-MS: 530.4 ([MþH]þ ).

4. Preparation of 1,4,7-Tris[(tert-butoxy)carbonyl]-10-{2-[(2-methylprop-2-enoyl)amino]acetyl}-1,4,7,10-tetraazacyclododecane (4). A dry THF soln. of 3 (1.06 g, 2.0 mmol) and Et3N (2.4 mmol) wascooled to �208. Methacryloyl chloride (2.4 mmol) was dissolved in 20 ml THF and added dropwise to thesoln. The resulting soln. was stirred at �208 for 0.5 h and then warmed to r.t. for 10 min. The suspensionwas filtered, and the precipitate was washed twice with a small amount of cold THF. The filtrate waspurified by CC (AcOEt /hexane; 2 :1) to give 4 (1.07 g, 91%). White amorphous solid. 1H-NMR(400 MHz, CDCl3): 5.79 (s, 1 H, ¼CH2); 5.37 (t, J¼2.4, 1 H, ¼CH2); 4.09–4.13 (m, CH2CO); 3.41–3.49(m, 8 CH2 (cyclen)); 1.99 (s, Me); 1.46 –1.48 (m, 9 Me (Boc)). HR-MS-ESI: 620.3647 ([MþNa]þ ,C29H51N5NaOþ

8 ; calc. 620.3630).5. Preparation of 1-{2-[(2-Methylprop-2-enoyl)amino]acetyl}-1,4,7,10-tetraazacyclododecane TFA

Salt (MAC· TFA; 5). To a soln. of 4 (898 mg, 1.5 mmol) in CH2Cl2 (0.5 ml), in a water-ice bath, TFA(0.5 ml) was added. The mixture was stirred at r.t. for 8 h. The mixture was then concentrated underreduced pressure below 408 to give the crude product. The resulting white oil was crystallized by anh.Et2O and washed three times with anh. Et2O (5 ml) to give a pale-white powder. 1H-NMR (400 MHz,D2O): 5.86 (s, 1 H, ¼CH2); 5.58 (s, 1 H, ¼CH2); 4.22 (s, CH2CO); 3.29 (s, 8 CH2 (cyclen)); 1.99 (s, Me).HR-MS-ESI: 292.2248 ([MþH]þ , C14H28N5Oþ

2 ; calc. 298.2238).6. Preparation of the Polymer PMAC (6). Compound 5 (500 mg, 1.7 mmol) was dissolved in H2O

(20 ml) and purged with N2 gas. After (NH4)2S2O8 (100 mg, 0.44 mmol)/NaHSO3 (75 mg, 0.72 mmol) wasdissolved in the mixture, the soln. was heated to 508 for 12 h. After concentration under reduced pressure,EtOH was added to obtain a pale-yellow ropy solid. This crude product was stirred in EtOH/aq. NaOH10 : 1 (v/v ; pH 11–12) for 10 h, and then soaked and washed with H2O until the pH 7.0, followed bywashing with EtOH for a few times, and the pale-yellow solid used in this study was obtained. IR (KBr):3421, 2880, 1683, 1202, 1128, 793, 721. 1H-NMR (400 MHz, D2O): 4.19 (s, CH2CO); 3.34–3.80 (m, CH2

(cyclen)); 1.94 (s, CH2); 1.24 (s, Me). GPC (Mw ¼108477; Mn¼67610; Mw/Mn¼1.60, around 365 cyclenunits in each polymer).

7. Fluorescence Quenching. Fluorescence quenching assay were performed in PBS buffer (pH 7.4,100 mm) at r.t. 10 ml of CT DNA (1 mg/ml) and 80 ml of EB (l mg/ml) were put into 3 ml of buffer in aquartz cuvette. Subsequently, the new further measurement was performed after mixing for a few min,when every PMAC soln. (1 mg/ml) was injected into the above buffer system.

8. DNA Release from the PMAC (6)/DNA Polyplex by NaPAA and NaCl. For DNA-release studies,sodium polyacrylate (NaPAA; high-molecular-weight polymer, Mw 8�106) and NaCl were used toinvestigate the polyplex stability. To the soln. of plasmid DNA in the presence of 6 (14.3 mg/l), variousconcentrations of NaPAA or NaCl were added, followed by dilution with the PBS buffer to a totalvolume of 17.5 ml, then it was incubated at 378 for 0.5 h, and the released plasmid DNA was analyzed byelectrophoresis on a 0.8% agarose gel.


9. Ge- Retardation Assay and CD Spectrum. PMAC aq. soln. of various concentrations were added tothe DNA soln. (25 mg/ml, 5 ml). NaH2PO4/Na2HPO4 (PBS) buffer was added to a final volume of 17.5 ml.Free DNA was used as control. The samples were briefly mixed prior to incubation at 378 for 30 min.DNA Loading buffer (1.5 ml) was added to the samples, which were then mixed again. Samples wereloaded into 0.8% agarose gel containing EB (1 mg/ml). Electrophoresis was carried out at 70 V in TAEbuffer (pH 7.40) for 60 min. The DNA band was visualized under a UV transilluminator. The CDspectrum was recorded when 10 mg/ml of Salmon sperm DNA was diluted to 1 mg/ml by using PBSbuffer (pH 7.40; 100 mm). Another CD spectrum was recorded after 25 ml of PMAC soln. (1 mg/ml) wasadded into 500 ml of the above buffer soln. of DNA.

10. Scanning Electron Microscopy (SEM) and Atomic Force Microscopy (AFM). The PMAC (6)soln. (10 ml, 0.1 mg/ml) was dropped into a clean glass in the absence or presence of pUC19 DNA (5 ml,25 mg/ml), and the sample was gently dried with clean air. The sample was disposed according to thestandard procedure and directly used for SEM test. Self-assembly of PMAC (6)/DNA polyplex wasstudied by tapping mode AFM. The pUC19 DNA (5 ml, 25 mg/ml) was treated with 6 (2.5 ml, 1.0 mg/ml) inPBS buffer (pH 7.40, 100 mm), followed by dilution with the corresponding PBS buffer to a total volumeof 17.5 ml. The final soln. was left to incubate at 378 for 30 min, and then it was diluted for 6000 times. 15 mlof sample soln. was blotted on a piece of freshly cleaved mica. Plasmids were left to be absorbed for ca.2 min at constant temp., and then the sample was gently rinsed with H2O and finally dried with clean air.Images were collected using a SPA400þ SPI3800N probe station operated in tapping mode in air at r.t.The noncontact silicon cantilevers used had a nominal radius of <20 nm and were driven at oscillationfrequencies in the range of 300 kHz. Images were simply flatted using the corresponding software, and nofurther processing was conducted.

11. Protection of PMAC (6)/DNA Polyplex against DNase I. The plasmid DNA solns. in the absenceand presence of 6 (14.3 mg/l) were separately incubated with 2U DNase I soln. (100 mm PBS buffer,pH 7.40) at 378 for 1 h, and degradation of plasmid DNA was analyzed by electrophoresis on a 0.8%agarose gel.

12. pH Titration Experiment. pH Measurements of HCl titrations of PMAC (6) soln. were performedwith a PHS-37C pH meter (0.01 pH unit sensitivity) and a E-201-C electrode. Titrations consisted ofincremental additions of 0.1n HCl into 5 ml of PMAC (6) soln. (1.0 mg/ml) in H2O. The buffer capacitywas calculated according to [33].

This work was financially supported by the National Science Foundation of China (Nos. 20725206,20732004, and 20572075), the Program for New Century Excellent Talents in University, the SpecializedResearch Fund for the Doctoral Program of Higher Education, and the Scientific Fund of SichuanProvince for Outstanding Young Scientist.

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Received February 25, 2008


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